The present invention relates to a method of reducing levels of TNF.alpha. and inhibiting phosphodiesterase in a mammal and to compounds and compositions useful therein.
TNF.alpha., or minor necrosis factor .alpha., is a cytokine which is released primarily by mononuclear phagocytes in response to various immunostimulators. When administered to animals or humans it causes inflammation, fever, cardiovascular effects, hemorrhage, coagulation and acute phase responses similar to those seen during acute infections and shock states.
Excessive or unregulated TNF.alpha. production has been implicated in a number of disease conditions. These include endotoxemia and/or toxic shock syndrome (Tracey et al., Nature 330, 662-664 (1987) and Hinshaw et al., Circ. Shock 30, 279-292 (1990)); cachexia (Dezube et al., Lancet, 335(8690), 662 (1990)); and Adult Respiratory Distress Syndrome where TNF.alpha. concentrations in excess of 12,000 pg/milliliters have been detected in pulmonary aspirates from ARDS patients (Millar et al., Lancet 2(8665), 712-714 (1989)). Systemic infusion of recombinant TNF.alpha. also resulted in changes typically seen in ARDS (Ferrai-Baliviera et al., Arch. Surg. 124(12), 1400-1405 (1989)).
TNF.alpha. appears to be involved in bone resorption diseases, including arthritis where it has been determined that when activated, leukocytes will produce a bone-resorbing activity, and data suggests that TNF.alpha. contributes to this activity (Bertolini et al., Nature 319, 516-518 (1986) and Johnson et al, Endocrinology 124(3), 1424-1427 (1989)). It has been determined that TNF.alpha. stimulates bone resorption and inhibits bone formation in vitro and in vivo through stimulation of osteoclast formation and activation combined with inhibition of osteoblast function. Although TNF.alpha. may be involved in many bone resorption diseases, including arthritis, the most compelling link with disease is the association between production of THF.alpha. by tumor or host tissues and malignancy associated hypercalcemia (Calci. Tissue Int. (US) 46(Suppl.), S3-10 (1990)). In Graft versus Host Disease, increased serun TNF.alpha. levels have been associated with major complications following acute allogenic bone marrow transplants (Holler et al., Blood, 75(4), 1011-1016 (1990)).
Cerebral malaria is a lethal hyperacute neurological syndrome associated with high blood levels of TNF.alpha. and is the most severe complication occurring in malaria patients. Levels of serum TNF.alpha. correlated directly with the severity of the disease and the prognosis in patients with acute malaria attacks (Grau et al., N. Engl. J. Med. 320(24), 1586-1591 (1989)).
TNF.alpha. also plays a role in the area of chronic pulmonary inflammatory diseases. The deposition of silica particles leads to silicosis, a disease of progressive respiratory failure caused by a fibrotic reaction. Antibodies to TNF.alpha. completely blocked the silica-induced lung fibrosis in mice (Pignet et al., Nature, 344:245-247 (1990)). High levels of TNF.alpha. production, in the serum and in isolated macrophages, have been demonstrated in animal models of silica and asbestos induced fibrosis (Bissonnette et al., Inflammation 13(3), 329-339 (1989)). Alveolar macrophages from pulmonary sarcoidosis patients have also been found to spontaneously release massive quantities of TNF.alpha. as compared with macrophages from normal donors (Baughman et al., J. Lab. Clin. Med. 115(1), 36-42 (1990)).
TNF.alpha. is also implicated in the inflammatory response which follows reperfusion, called reperfusion injury, and is a major cause of tissue damage after loss of blood flow (Vedder et al., PNAS 87, 2643-2646 (1990)). TNF.alpha. also alters the properties of endothelial cells and has various pro-coagulant activities, such as producing an increase in tissue factor pro-coagulant activity and suppression of the anticoagulant protein C pathway as well as down-regulating the expression of thrombomodulm (Sherry et al., J. Cell Biol. 107, 1269-1277 (1988)). TNF.alpha. has pro-inflammatory activities which together with its early production (during the initial stage of an inflammatory event) make it a likely mediator of tissue injury in several important disorders including but not limited to, myocardial infarction, stroke and circulatory shock. Of specific importance may be TNF.alpha.-induced expression of adhesion molecules, such as intercellular adhesion molecule (ICAM) or endothelial leukocyte adhesion molecule (ELAM) on endothelial cells (Munro et al., Am. J. Path. 135(1), 121-132 (1989)).
Moreover, it is now known that TNF.alpha. is a potent activator of retrovirus replication including activation of HIV-1. (Duh et al., Proc. Nat. Acad. Sci. 86, 5974-5978 (1989); Poll et al., Proc. Nat. Acad. Sci. 87, 782-785 (1990); Monto et al., Blood 79, 2670 (1990); Clouse et al., J. Immunol. 142, 431-438 (1989); Poll et al., AIDS Res. Hum. Retrovirus, 191-197 (1992)). AIDS results from the infection of T lymphocytes with Human Immunodeficiency Virus (HIV). At least three types or strains of HIV have been identified, i.e., HIV-1, HIV-2 and HIV-3. As a consequence of HIV infection, T-cell mediated immunity is impaired and infected individuals manifest severe opportunistic infections and/or unusual neoplasms. HIV entry into the T lymphocyte requires T lymphocyte activation. Other viruses, such as HIV-1 and HIV-2 infect T lymphocytes after T cell activation and such virus protein expression and/or replication is mediated or maintained by such T cell activation. Once an activated T lymphocyte is infected with HIV, the T lymphocyte must continue to be maintained in an activated state to permit HIV gene expression and/or HIV replication. Cytokines, specifically TNF.alpha., are implicated in activated T-cell mediated HIV protein expression and/or virus replication by playing a role in maintaining T lymphocyte activation. Therefore, interference with cytokine activity such as by prevention, control, or inhibition of cytokine production, notably TNF.alpha., in a HIV-infected individual aids in limiting the maintenance of T lymphocyte activation caused by HIV infection.
Monocytes, macrophages, and related cells, such as kupffer and glial cells, have also been implicated in maintenance of the HIV infection. These cells, like T cells, are targets for viral replication and the level of viral replication is dependent upon the activation state of the cells (Rosenberg et al., The Immunopathogenesis of HIV Infection, Advances in Immunology, 57 (1989)). Cytokines, such as TNF.alpha., have been shown to activate HIV replication in monocytes and/or macrophages (Poli et al., Proc. Natl. Acad. Sci., 87, 782-784 (1990)), therefore, prevention, control, or inhibition of cytokine production or activity aids in limiting HIV progression as stated above for T cells. Additional studies have identified TNF.alpha. as a common factor in the activation of HIV in vitro and has provided a clear mechanism of action via a nuclear regulatory protein found in the cytoplasm of cells (Osborn, et al., PNAS 86, 2336-2340). This evidence suggests that a reduction of TNF.alpha. synthesis may have an antiviral effect in HIV infections, by reducing the transcription and thus virus production.
HIV viral replication of latent HIV in T cell and macrophage lines can be induced by TNF.alpha.(Folks et al., PNAS 86, 2365-2368 (1989)). A molecular mechanism for the virus inducing activity is suggested by TNF.alpha.'s ability to activate a gene regulatory protein (NF.kappa.B) found in the cytoplasm of cells, which promotes HIV replication through binding to a viral regulatory gene sequence (LTR) (Osborn et al., PNAS 86, 2336-2340 (1989)). TNF.alpha. in AIDS associated cachexia is suggested by elevated serum TNF.alpha. and high levels of spontaneous TNF.alpha. production in peripheral blood monocytes from patients (Wright et al. J. Immunol. 141(1), 99-104 (1988)).
TNF.alpha. has been implicated in various roles with other viral infections, such as the cytomegalia virus (CMV), influenza virus, adenovirus, and the herpes family of viruses for similar reasons as those noted.
Preventing, controlling, or inhibiting the production or action of TNF.alpha. (e.g. treatment with the compounds of this invention) is, therefore, predicted to be a potent therapeutic strategy for many inflammatory, infectious, immunological or malignant diseases. These include but are not restricted to septic shock, sepsis, endotoxic shock, hemodynamic shock and sepsis syndrome, post ischemic reperfusion injury, malaria, mycobacterial infection, meningitis, psoriasis, congestive heart failure, fibrotic disease, cachexia, graft rejection, cancer, autoimmune disease, opportunistic infections in AIDS, rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, other arthritic conditions, Crohn's disease, ulcerative colitis, multiple sclerosis, systemic lupus erythrematosis, ENL in leprosy, radiation damage, asthma, and hyperoxic alveolar injury. Efforts directed to the suppression of the effects of TNF.alpha. have ranged from the utilization of steroids such as dexamethasone and prednisolone to the use of both polyclonal and monoclonal antibodies (Beutler et al., Science 234, 470-474 (1985); WO 92/11383).
The nuclear factor .kappa.B (NF.kappa.B) is a pleiotropic transcriptional activator (Lenardo, et al. Cell 1989, 58, 227-29). NF.kappa.B has been implicated as a transcriptional activator in a variety of disease and inflammatory states and is thought to regulate cytokine levels including but not limited to TNF.alpha. and also to be an activator of HIV transcription (Dbaibo, et al. J. Biol. Chem. 1993, 17762-66; Duh et al. Proc. Natl. Acad. Sci. 1989, 86, 5974-78; Bachelerie et al. Nature 1991, 350, 709-12; Boswas, J. et al. Acquired Immune Deficiency Syndrome 1993, 6, 778-786; Suzuki et al. Biochem. And Biophys. Res. Comm. 1993, 193, 277-83; Suzuki et al. Biochem. And Biophys. Res Comm. 1992, 189, 1709-15; Suzuki et al. Biochem. Mol. Bio. Int. 1993, 31(4), 693-700; Shakhov et al. 1990, 171, 35-47; and Staal et al. Proc. Natl. Acad. Sci. USA 1990, 87, 9943-47). Thus, inhibition or activation of NF.kappa.B binding can regulate transcription of cytokine gene(s) and through this modulation and other mechanisms be useful in the inhibition of a multitude of disease states. The compounds claimed in this patent can inhibit the action of NF.kappa.B in the nucleus and thus are useful in the treatment of a variety of diseases including but not limited to asthma, rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, other arthritic conditions, septic shock, septis, endotoxic shock, graft versus host disease, wasting, Crohn's disease, ulcerative colitis, multiple sclerosis, systemic lupus erythrematosis, ENL in leprosy, HIV, AIDS, and opportunistic infections in AIDS.
TNF.alpha. and NF.kappa.B levels are influenced by a reciprocal feedback loop. As noted above, the compounds of the present invention affect the levels of both TNF.alpha. and NF.kappa.B. It is not known at this time, however, how the compounds of the present invention regulate the levels of TNF.alpha., NF.kappa.B, or both.
Many cellular functions can be mediated by levels of adenosine 3',5'-cyclic monophosphate(cAMP). Such cellular functions can contribute to inflammatory conditions and diseases including asthma, inflammation, and other conditions (Lowe and Cheng, Drugs of the Future, 17(9), 799-807, 1992). It has been shown that the elevation of cAMP in inflammatory leukocytes inhibits their activation and the subsequent release of inflammatory mediators. Increased levels of cAMP also leads to the relaxation of airway smooth muscle.
The primary cellular mechanism for the inactivation of cAMP is the breakdown of cAMP by a family of isoenzymes referred to as cyclic nucleotide phosphodiesterases (PDE) (Beavo and Reitsnyder, Trends in Pharm., 11, 150-155, 1990). There are seven known members of the family of PDEs. It is recognized, for example, that the inhibition of PDE type IV is particularly effective in both the inhibition of inflammatory mediator release and the relaxation of airway smooth muscle (Verghese, et al., Journal of Pharmacology and Experimental Therapeutics, 272(3), 1313-1320, 1995). Thus, compounds that inhibit PDE IV specifically, would exhibit the desirable inhibition of inflammation and relaxation of airway smooth muscle with a minimum of unwanted side effects, such as carclio-vascular or anti-platelet effects. Currently used PDE IV inhibitors lack the selective action at acceptable therapeutic doses.
The compounds of the present invention are useful in the inhibition of phosphodiesterases, particularly PDE III and PDE IV, and in the treatment of disease states mediated thereby.